Journal: Development (Cambridge, England)

Article Title: Proteomic profile of hippocampal growth cones through early postnatal development

doi: 10.1242/dev.205342

Figure Lengend Snippet: Invasion of the hippocampus by the fibres of entorhinal cortex reelin-positive Layer 2 cells. (A) Confocal image of horizontal sections of the hippocampus (HP) of Odz3-tTa;TetO-GCamP6 animals at P1, P3 and P5. NeuN staining (turquoise) is used to label neuronal cell bodies. Staining of the GFP part of GCamP6 (GFP) labels fibres of reelin-positive Layer 2 cells (yellow). Dashed line indicates the hippocampal fissure. (B) Quantification of the increase in GFP fluorescent signal in the stratum lacunosum-moleculare of cornu ammonis 3 (CA3 SLM) and in the molecular layer (ML) of the inner and outer blade of the dentate gyrus (DG) over time reflecting how the invasion progresses during the early postnatal days. Depicted is the normalized volume of fluorescent voxels over the background threshold for each subregion of interest. Datapoints identified as outliers (quartile method) are depicted as crosses. Inlay shows schematic of quantified areas (CA3 SLM in turquoise, inner blade DG in green, outer blade DG in pink) and a represented delineated image for quantification is shown in Fig. S1B . (C) Representative confocal images of growth cones (GCs) of reelin-positive Layer 2 cells in CA3 SLM at P1 (left), inner ML at P3 (middle) and outer ML at P5 (right). GCs are indicated by Stx7 puncta (magenta) at the end of GFP-positive fibres (yellow); see arrows. (D) Quantification of GC density. Bar plots show normalized mean and standard deviation of Stx7+GFP-positive events for each delineated area. Three non-consecutive slices of three different animals were counted for the Stx7+GFP events for each timepoint ( n =9). Given numbers are normalized to total counts per slice. Scale bars: 200 μm (A); 5 μm (C).

Article Snippet: The slides were then incubated overnight at 4°C with the following primary antibodies: NeuN (guinea pig, anti-NeuN, Sigma Millipore, #ABN90P, 1:1000), GFP in GCamP6 (chicken, anti-GFP, Abcam, #AB13970, 1:1000; to detect GFP part of GCamP6) and Stx7 (rabbit, anti-Stx7, Synaptic Systems, #110072, 1:200).

Techniques: Staining, Standard Deviation

Journal: The EMBO Journal

Article Title: SNARE mimicry by the CD225 domain of IFITM3 enables regulation of homotypic late endosome fusion

doi: 10.1038/s44318-024-00334-8

Figure Lengend Snippet: ( A ) Human endosomal SNARE proteins were aligned with human IFITM proteins, and a 53-residue stretch of sequence is shown. Residues in red text are the central polar arginine or glutamine residues characteristic of canonical SNARE proteins and are labeled as the central “0” layer. Surrounding heptad repeats of hydrophobic residues are labeled from “−7” to “+8” in relation to the “0” layer, and sites highlighted in yellow correspond to hydrophobic residues. Black underlined residues in IFITM3 were mutated for functional experiments. Green underlined residues in IFITM3 correspond to the previously described amphipathic alpha helix. ( B ) HEK293T cells were transfected with IFITM3-FLAG (WT, F75/78A, R85Q, or G95L) and, 48 h later, challenged with IAV (A/PR/8/34 (PR8), H1N1) at a multiplicity of infection of 0.10. 18 h post-infection, cells were fixed, permeabilized, immunostained with anti-FLAG and anti-NP, and analyzed by flow cytometry. The percentage of NP+ cells was measured as a fraction of FLAG+ cells and shown as mean and standard error (normalized relative to Empty Vector, which was set to 100%). Infections were performed independently three times (biological replicates). Differences that were statistically significant from WT as determined by one-way ANOVA are indicated by (*). Exact p values are as follows (from left to right): p < 0.0001, p < 0.0001, p < 0.0001. ( C ) Structural prediction of the R-SNARE-like motif of IFITM3 was performed with Alphafold and FATCAT. Residues 55–113 of IFITM3 were modeled against a template of the R-SNARE motif of VAMP8 which was previously crystallized as part of the STX7-STX8-Vti1b-VAMP8 trans-SNARE complex (PDB: 1GL2). ( D ) Left: HEK293T cells were co-transfected with IFITM3-FLAG or Empty Vector and a construct encoding a single SNARE protein: STX7-HA, STX8-HA, Vti1b-HA, VAMP8-HA, or VAMP7-myc. SDS-PAGE and immunoblotting were performed with anti-HA, anti-myc, and anti-FLAG in whole cell lysates. Anti-actin was used as loading control. Right: From co-transfected cells, IFITM3-FLAG was immunoprecipitated with anti-FLAG followed by SDS-PAGE and immunoblotting with anti-HA, anti-myc, and anti-FLAG. Heavy chain immunoglobulin chain was used as loading control. Co-transfection of Empty Vector and STX7-HA was used as a negative control for immunoprecipitation. ( E ) Left: HEK293T cells were co-transfected with STX7-HA and either IFITM3-FLAG (WT, F75/78A, or R85Q) or Empty Vector. SDS-PAGE and immunoblotting were performed with anti-HA and anti-FLAG in whole cell lysates. Anti-actin was used as loading control. Right: From co-transfected cells, IFITM3-FLAG was immunoprecipitated with anti-FLAG followed by SDS-PAGE and immunoblotting with anti-FLAG and anti-HA. Light chain immunoglobulin was used as loading control. Co-transfection of Empty Vector and STX7-HA was used as a negative control for immunoprecipitation. The HA/FLAG ratio was calculated for the indicated lanes and shown as mean and standard error (normalized relative to WT, which was set to 100%). Differences that were statistically significant from WT as determined by one-way ANOVA are indicated by (*). Exact p values are as follows (from left to right): p < 0.0001, p < 0.0001. Numbers and tick marks left of blots indicate position and size (in kilodaltons) of protein standard in ladder. Immunoblots were performed independently three times, and a representative example is shown. IAV Influenza A virus, Ig immunoglobulin, IP immunoprecipitation, EV Empty Vector, WT wild-type. .

Article Snippet: The samples were incubated with 1 µg of anti-STX7 antibody for 1 h. The antibody-antigen complex was captured with 15 µL of Dynabeads (Invitrogen).

Techniques: Residue, Sequencing, Labeling, Functional Assay, Transfection, Infection, Flow Cytometry, Plasmid Preparation, Construct, SDS Page, Western Blot, Control, Immunoprecipitation, Cotransfection, Negative Control, Virus

Journal: The EMBO Journal

Article Title: SNARE mimicry by the CD225 domain of IFITM3 enables regulation of homotypic late endosome fusion

doi: 10.1038/s44318-024-00334-8

Figure Lengend Snippet: ( A ) The R-SNARE-like motif of IFITM3 and the R-SNARE motif of VAMP8 (53 residues each) were compared by predictive protein structure alignment and comparison algorithm TM-align. A TM score of 0.75847 was recorded, indicating that the two regions are likely to adopt the same alpha helical protein fold. ( B ) Structural prediction of the R-SNARE-like motif of IFITM3 was performed with Alphafold and FATCAT. Residues 55–113 of IFITM3 were modeled against a template of the R-SNARE motif of VAMP8 which was previously crystallized as part of the STX7-STX8-Vti1b-VAMP8 trans-SNARE complex (PDB: 1GL2). VAMP8 was then swapped with the predicted structure of IFITM3 and shown in relation to the coiled-coiled structure formed with STX7, STX8, and Vti1b. Top: the side chains of residues F75, F78, and R85 of IFITM3 are depicted in yellow and labeled. Bottom: alternative view of the side chains of F75, F78, and R85 of IFITM3. ( C ) Top: examination of the central polar “0” layer of the Q-SNAREs STX7, STX8, Vti1b in relation to the R85 residue in the predicted configuration of IFITM3. Bottom: examination of the central polar “0” layer of the STX7-STX8-Vti1b-VAMP8 complex (PDB: IGL2). .

Article Snippet: The samples were incubated with 1 µg of anti-STX7 antibody for 1 h. The antibody-antigen complex was captured with 15 µL of Dynabeads (Invitrogen).

Techniques: Comparison, Labeling, Residue

Journal: The EMBO Journal

Article Title: SNARE mimicry by the CD225 domain of IFITM3 enables regulation of homotypic late endosome fusion

doi: 10.1038/s44318-024-00334-8

Figure Lengend Snippet: ( A ) Top: HEK293T cells were co-transfected with STX8-HA, STX7-HA, or Vti1b-HA and either Empty Vector or IFITM3-FLAG (WT, F75/78A, or G95L). SDS-PAGE and immunoblotting were performed with anti-HA and anti-FLAG in whole cell lysates. Anti-tubulin was used as loading control. Bottom: from co-transfected cells, STX8-HA, STX7-HA, or Vti1b-HA were immunoprecipitated with anti-HA followed by SDS-PAGE and immunoblotting with anti-HA and anti-FLAG. Heavy chain immunoglobulin was used as loading control. Co-transfection of Empty Vector and STX8-HA was used as a negative control for immunoprecipitation. The FLAG/HA ratio was calculated for the indicated lanes and shown as mean and standard error (normalized relative to WT with STX7-HA, which was set to 100%). Differences that were statistically significant from WT with STX7-HA as determined by one-way ANOVA are indicated by (*). Exact p values are as follows (from left to right): p < 0.0001, p < 0.0001, p < 0.0001, p < 0.0001, p < 0.0001. Numbers and tick marks left of blots indicate position and size (in kilodaltons) of protein standard in ladder. Immunoblots were performed independently three times, and a representative example is shown. ( B ) HEK293T cells stably expressing Empty Vector, IFITM3 WT-FLAG, or IFITM3 G95L-FLAG were transfected with STX7-GFP, fixed, immunostained with anti-FLAG, and analyzed by confocal immunofluorescence microscopy. Colocalization was measured between FLAG and STX7-GFP by calculating the Pearson’s correlation coefficient using Fiji software. Coefficients were calculated from medial Z-slices from three fields of view containing 5–15 cells per condition and presented as means and standard error. Scale bar = 15 microns. Ig immunoglobulin, IP immunoprecipitation, EV Empty Vector, WT wild-type, PCC Pearson’s correlation coefficient. .

Article Snippet: The samples were incubated with 1 µg of anti-STX7 antibody for 1 h. The antibody-antigen complex was captured with 15 µL of Dynabeads (Invitrogen).

Techniques: Transfection, Plasmid Preparation, SDS Page, Western Blot, Control, Immunoprecipitation, Cotransfection, Negative Control, Stable Transfection, Expressing, Immunofluorescence, Microscopy, Software

Journal: The EMBO Journal

Article Title: SNARE mimicry by the CD225 domain of IFITM3 enables regulation of homotypic late endosome fusion

doi: 10.1038/s44318-024-00334-8

Figure Lengend Snippet: ( A ) Left: HeLa cells (WT or IFITM3 KO) were transfected with STX7-HA. SDS-PAGE and immunoblotting were performed with anti-HA and anti-IFITM3 in whole cell lysates. Anti-actin was used as loading control. Right: STX7-HA was immunoprecipitated with anti-HA antibody followed by SDS-PAGE and immunoblotting with anti-HA and anti-IFITM3. Heavy chain immunoglobulin was used as loading control. An isotype matched antibody was used as a control for immunoprecipitation. ( B ) Endogenous STX8 was immunoprecipitated from HeLa (WT or IFITM3 KO) with anti-STX8 followed by SDS-PAGE and immunoblotting with anti-STX8 and anti-IFITM3. Heavy chain immunoglobulin was used as loading control. An isotype matched antibody was used as a control for immunoprecipitation. The (#) symbol denotes the presence of light chain immunoglobulin. ( C ) Endogenous Vti1b was immunoprecipitated from HeLa (WT or IFITM3 KO) with anti-Vti1b followed by SDS-PAGE and immunoblotting with anti-Vti1b and anti-IFITM3. Light chain immunoglobulin was used as loading control. ( D ) Endogenous VAMP8 was immunoprecipitated from HeLa (WT or IFITM3 KO) with anti-VAMP8 followed by SDS-PAGE and immunoblotting with anti-VAMP8 and anti-IFITM3. Light chain immunoglobulin was used as loading control. ( E ) Endogenous VAMP7 was immunoprecipitated from HeLa (WT or IFITM3 KO) with anti-VAMP7 followed by SDS-PAGE and immunoblotting with anti-VAMP7 and anti-IFITM3. Two forms of VAMP7 were detected following immunoprecipitation: one form of ~25 kD and another of ~15 kD (Wojnacki et al, ). Light chain immunoglobulin was used as loading control. ( F ) Left: Recombinant STX7 and recombinant IFITM3 (WT, F75/78A, or G95L) were mixed together in vitro and reaction inputs were visualized by SDS-PAGE and immunoblotting with anti-STX7 and anti-IFITM3. Right: recombinant STX7 was immunoprecipitated with anti-STX7 followed by SDS-PAGE and immunoblotting with anti-STX7 and anti-IFITM3. Heavy chain immunoglobulin was used as loading control. Numbers and tick marks left of blots indicate position and size (in kilodaltons) of protein standard in ladder. Immunoblots were performed independently two times and a representative example is shown. Ig immunoglobulin, IP immunoprecipitation, KO knockout, WT wild-type, r recombinant. .

Article Snippet: The samples were incubated with 1 µg of anti-STX7 antibody for 1 h. The antibody-antigen complex was captured with 15 µL of Dynabeads (Invitrogen).

Techniques: Transfection, SDS Page, Western Blot, Control, Immunoprecipitation, Recombinant, In Vitro, Knock-Out

Journal: The EMBO Journal

Article Title: SNARE mimicry by the CD225 domain of IFITM3 enables regulation of homotypic late endosome fusion

doi: 10.1038/s44318-024-00334-8

Figure Lengend Snippet: ( A ) HeLa cells (WT or IFITM3 KO) were subjected to whole cell lysis, SDS-PAGE, and immunoblotting with anti-IFITM2/3, anti-STX8, anti-Vti1b, anti-VAMP8, anti-VAMP7, and anti-actin. Red and green arrows indicate IFITM3 and IFITM2, respectively, recognized by the anti-IFITM2/3 antibody. ( B ) STX7-HA was transfected into HeLa (WT or IFITM3 KO) and immunoprecipitated with anti-HA followed by SDS-PAGE and immunoblotting with anti-HA, anti-IFITM3, anti-STX8, and anti-Vti1b. Heavy chain immunoglobulin was used as loading control. An isotype matched antibody was used as a control for immunoprecipitation. The (#) symbol denotes the presence of light chain immunoglobulin. ( C ) Purified recombinant IFITM3 protein (WT, F75/78A, or G95L) of varying inputs (6, 12, or 18 µg) were subjected to SDS-PAGE and visualized by Coomassie stain. ( D ) 20 ng of purified recombinant IFITM3 protein (WT, F75/78A, or G95L) were subjected to SDS-PAGE and immunoblotting with anti-IFITM3. ( E ) Recombinant STX7 of varying inputs (0.5, 1, or 2 µg) were subjected to SDS-PAGE and visualized by Coomassie stain. ( F ) Recombinant STX7 was subjected to SDS-PAGE and immunoblotting with anti-STX7. Numbers and tick marks left of blots indicate position and size (in kilodaltons) of protein standard in ladder. Immunoblots were performed independently two times and a representative example is shown (for ( A ) and ( B )) or once (for ( C ), ( D ), ( E ), and ( F )). Ig immunoglobulin, IP immunoprecipitation, EV Empty Vector, WT wild-type, r recombinant. .

Article Snippet: The samples were incubated with 1 µg of anti-STX7 antibody for 1 h. The antibody-antigen complex was captured with 15 µL of Dynabeads (Invitrogen).

Techniques: Lysis, SDS Page, Western Blot, Transfection, Immunoprecipitation, Control, Purification, Recombinant, Staining, Plasmid Preparation

Journal: The EMBO Journal

Article Title: SNARE mimicry by the CD225 domain of IFITM3 enables regulation of homotypic late endosome fusion

doi: 10.1038/s44318-024-00334-8

Figure Lengend Snippet: ( A ) Size exclusion chromatography was performed on recombinant IFITM3 proteins (WT, F75/78A, and G95L) and eluted fractions were analyzed by SDS-PAGE and Coomassie staining. 150 µg recombinant protein was loaded as input and fraction numbers correspond to mL volumes eluted from column. ( B ) Left: HEK293T cells stably expressing Empty Vector, IFITM3 WT-FLAG, or IFITM3 G95L-FLAG were co-transfected with STX7-HA and VAMP7-Myc. Whole cell lysates were subjected to SDS-PAGE and immunoblotting with anti-HA, anti-FLAG, and anti-Myc. Actin was used as a loading control. Right: STX7-HA was immunoprecipitated with anti-HA followed by SDS-PAGE and immunoblotting with anti-HA, anti-FLAG, and anti-Myc. The Myc/HA ratio was calculated for the indicated lanes and shown as mean and standard error (normalized relative to Empty Vector, which was set to 100%). Differences that were statistically significant from Empty Vector as determined by one-way ANOVA are indicated by (*). Exact p values are as follows (from left to right): p = 0.0099, p = 0.8782. Numbers and tick marks left of blots indicate position and size (in kilodaltons) of protein standard in ladder. Immunoblots were performed independently three times, and a representative example is shown (except ( A ), which was performed once). ( C ) HeLa (WT or IFITM3 KO) were transfected with STX7-GFP and VAMP8-mCherry and confocal immunofluorescence microscopy was performed. Colocalization between STX7-GFP and VAMP8-mCherry was measured by calculating the Pearson’s correlation coefficient using Fiji software. Coefficients were calculated from medial Z-slices from three fields of view containing 5–15 cells per condition and presented as means and standard error. Scale bar = 15 microns. IP immunoprecipitation, WT wild-type, r recombinant. .

Article Snippet: The samples were incubated with 1 µg of anti-STX7 antibody for 1 h. The antibody-antigen complex was captured with 15 µL of Dynabeads (Invitrogen).

Techniques: Size-exclusion Chromatography, Recombinant, SDS Page, Staining, Stable Transfection, Expressing, Plasmid Preparation, Transfection, Western Blot, Control, Immunoprecipitation, Immunofluorescence, Microscopy, Software

Journal: The EMBO Journal

Article Title: SNARE mimicry by the CD225 domain of IFITM3 enables regulation of homotypic late endosome fusion

doi: 10.1038/s44318-024-00334-8

Figure Lengend Snippet: ( A ) HEK293T cells stably expressing Empty Vector, IFITM3 WT-FLAG, or IFITM3 G95L-FLAG were co-transfected with STX7-HA and VAMP8-Myc. Whole cell lysates were subjected to SDS-PAGE and immunoblotting with anti-HA, anti-FLAG, and anti-Myc. Actin was used as a loading control. Right: STX7-HA was immunoprecipitated with anti-HA followed by SDS-PAGE and immunoblotting with anti-HA, anti-FLAG, and anti-Myc. The Myc/HA ratio was calculated for the indicated lanes and shown as mean and standard error (normalized relative to Empty Vector, which was set to 100%). Differences that were statistically significant from Empty Vector as determined by one-way ANOVA are indicated by (*). Exact p values are as follows (from left to right): p = 0.0016, p = 0.4318. ( B ) HEK293T cells stably expressing Empty Vector, IFITM3 WT-FLAG, or IFITM3 G95L-FLAG were transfected with STX7-GFP and VAMP8-mCherry, fixed, and analyzed by confocal immunofluorescence microscopy. Colocalization was measured between STX7-GFP and VAMP8-mCherry by calculating the Pearson’s correlation coefficient using Fiji software. Coefficients were calculated from medial Z-slices from three fields of view containing 5–15 cells per condition and presented as means and standard error. Scale bar = 15 microns. ( C ) HEK293T cells stably expressing Empty Vector, IFITM3 WT-FLAG, or IFITM3 G95L-FLAG were inoculated with IAV (+) or medium (−) for 18 h and subjected to whole cell lysis. Endogenous STX8 was immunoprecipitated with anti-STX8 followed by SDS-PAGE and immunoblotting with anti-STX8, anti-FLAG, anti-VAMP8, and anti-VAMP7. The VAMP8/STX8 and VAMP7/STX8 ratios were calculated for the indicated lanes and shown as mean and standard error (normalized relative to No Virus Empty Vector, which was set to 100%). Differences that were statistically significant from No Virus Empty Vector as determined by one-way ANOVA are indicated by (*). Exact p values are as follows (from left to right, top to bottom): p = 0.0007, p = 0.6986, p = 0.0093, p = 0.4523, p < 0.0001, p = 0.7960. ( D ) Endogenous STX8 was immunoprecipitated from HeLa cells (WT or IFITM3 KO) with anti-STX8 followed by SDS-PAGE and immunoblotting with anti-STX8, anti-IFITM3, and anti-VAMP8. Light chain immunoglobulin was used as loading control. An isotype matched antibody was used as a control for immunoprecipitation. The VAMP8/STX8 ratio was calculated for the indicated lanes and shown as mean and standard error (normalized relative to WT, which was set to 100%). Differences that were statistically significant from WT as determined by student’s T test are indicated by (*). Exact p value: p = 0.0009. Numbers and tick marks left of blots indicate position and size (in kilodaltons) of protein standard in ladder. The (#) symbol denotes the presence of light chain immunoglobulin. Immunoblots were performed independently three times, and a representative example is shown. Ig immunoglobulin, IP immunoprecipitation, EV Empty Vector, WT wild-type, PCC Pearson’s correlation coefficient. .

Article Snippet: The samples were incubated with 1 µg of anti-STX7 antibody for 1 h. The antibody-antigen complex was captured with 15 µL of Dynabeads (Invitrogen).

Techniques: Stable Transfection, Expressing, Plasmid Preparation, Transfection, SDS Page, Western Blot, Control, Immunoprecipitation, Immunofluorescence, Microscopy, Software, Lysis, Virus

Journal: The EMBO Journal

Article Title: SNARE mimicry by the CD225 domain of IFITM3 enables regulation of homotypic late endosome fusion

doi: 10.1038/s44318-024-00334-8

Figure Lengend Snippet: ( A ) HEK293T stably expressing Empty Vector, IFITM3 WT-FLAG, or IFITM3 G95L-FLAG were co-transfected with STX7-HA, STX8-HA, Vti1b-HA, and VAMP8-Myc. Following whole cell lysis, samples were either boiled at 100 °C (+) or not (−). SDS-PAGE and immunoblotting was performed with anti-Myc and anti-FLAG. Actin was used as loading control. Numbers and tick marks left of blots indicate position and size (in kilodaltons) of protein standard in ladder. Immunoblots were performed independently twice, and a representative example is shown. ( B ) HEK293T cells stably expressing Empty Vector, IFITM3 WT-FLAG, or IFITM3 G95L-FLAG were pulsed with Dextran Alexa Fluor 488 for 2 h followed by addition of Magic Red for 5 min. Living cells were then analyzed immediately by confocal immunofluorescence microscopy. Colocalization between Dextran and Magic Red was measured by calculating the Pearson’s correlation coefficient using Fiji software. Coefficients were calculated from medial Z-slices from three fields of view containing 5–15 cells per condition and presented as means and standard error. Scale bar = 15 microns. PCC Pearson’s correlation coefficient. .

Article Snippet: The samples were incubated with 1 µg of anti-STX7 antibody for 1 h. The antibody-antigen complex was captured with 15 µL of Dynabeads (Invitrogen).

Techniques: Stable Transfection, Expressing, Plasmid Preparation, Transfection, Lysis, SDS Page, Western Blot, Control, Immunofluorescence, Microscopy, Software

Journal: The EMBO Journal

Article Title: SNARE mimicry by the CD225 domain of IFITM3 enables regulation of homotypic late endosome fusion

doi: 10.1038/s44318-024-00334-8

Figure Lengend Snippet: IFITM3 selectively regulates the trans-SNARE assembly driving late endosome-late endosome fusion (STX7-STX8-Vti1b-VAMP8), while enabling late endosome-lysosome fusion. As a result, endocytic cargos (including viruses) are more efficiently trafficked to lysosomes in IFITM3-expressing cells. LE late endosome, Lys lysosome.

Article Snippet: The samples were incubated with 1 µg of anti-STX7 antibody for 1 h. The antibody-antigen complex was captured with 15 µL of Dynabeads (Invitrogen).

Techniques: Expressing

Journal: The EMBO Journal

Article Title: SNARE mimicry by the CD225 domain of IFITM3 enables regulation of homotypic late endosome fusion

doi: 10.1038/s44318-024-00334-8

Figure Lengend Snippet: Reagents and tools table

Article Snippet: The samples were incubated with 1 µg of anti-STX7 antibody for 1 h. The antibody-antigen complex was captured with 15 µL of Dynabeads (Invitrogen).

Techniques: Recombinant, Plasmid Preparation, Sequencing, Protease Inhibitor, Immunoprecipitation, Virus, Software

Journal: The EMBO Journal

Article Title: SNARE mimicry by the CD225 domain of IFITM3 enables regulation of homotypic late endosome fusion

doi: 10.1038/s44318-024-00334-8

Figure Lengend Snippet: Reagents and tools table

Article Snippet: The following antibodies were used in this study: anti-STX8 (110-083; Synaptic Systems); anti-STX7 (110-072; Synaptic Systems), anti-Vti1b (164-002; Synaptic Systems), anti-VAMP8 (104-302; Synaptic Systems), anti-VAMP7 (ab36195; Abcam), anti-HA (901514, clone 16B12; Biolegend), anti-HA (ab9110; Abcam (this was used for immunoprecipitation)), anti-Myc (C3956, Sigma), anti-FLAG M2 (F1804; Sigma), anti-IFITM3 (ab109429; Abcam), anti-IFITM2/3 (66081-1-Ig, Proteintech), anti-tubulin and anti-actin (SC-47778; Santa Cruz Biotechnology).

Techniques: Recombinant, Plasmid Preparation, Sequencing, Protease Inhibitor, Immunoprecipitation, Virus, Software

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Identification of the role of SNARE proteins in rAAV vector production through interaction with the viral MAAP

doi: 10.1016/j.omtm.2024.101392

Figure Lengend Snippet: STX7 or SNAP23 colocalizes with MAAP2 in both wtAAV2-infected cells and rAAV2-producing cells (A and D) wtAAV2 infection. HEK293 cells were mock-infected or infected with wtAAV2 followed by pHelper transfection. At 2 dpi, the cells were co-immunostained for MAAP2 and SNAP23 (A) or MAAP2 and STX7 (C). (B and E) rAAV2 production. HEK293 cells were mock or transfected with pR2C2, pHelper, and prAAV2. At 2 dpt, the transfected cells were co-immunostained for MAAP2 and SNAP23 (B) or MAAP2 and STX7 (D). SNAP23 (B) or STX7 (D) cells were co-immunostained with a secondary antibody conjugated with a far-red dye. Co-immunostained cells were observed under a Leica STED microscope with a 100× objective lens. Images captured in the far-red wavelength were pseudo-colored in red. The colors of confocal images correspond to blue for DAPI, green for MAAP, and red for SNAP23 or STX7 as indicated. Scale bar, 5 μm. Representative confocal images are shown. (C and F) Quantification of colocalization. Pearson’s correlation coefficients were measured for colocalization of MAAP2 with SNAP23 (C) or STX7 (F) using NIH ImageJ.

Article Snippet: An anti-V5 (#R960-25) was purchased from Invitrogen (Carlsbad, CA), and anti-STX7 antibodies were purchased from AbClonal (#A8057) and Bethyl Laboratories (#A304-512A; Montgomery, TX) for immunostaining and immunoblotting, respectively.

Techniques: Infection, Transfection, Microscopy

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Identification of the role of SNARE proteins in rAAV vector production through interaction with the viral MAAP

doi: 10.1016/j.omtm.2024.101392

Figure Lengend Snippet: SNAP23 or STX7 interacts with MAAP2 in cells but not in vitro (A and B) Co-IP. HEK293 cells were transfected with pCI-MAAP2 Flag or pCI-empty. At 2 dpt, cells were harvested and lysed. 90% of the lysates were used for immunoprecipitation with anti-Flag-conjugated magnetic beads. Western blotting was performed for detection of MAAP2 Flag and SNAP23, respectively (A), and for detection of MAAP2 Flag and STX7, respectively (B). β-actin is shown as a loading control. 10% of the lysates were loaded as the whole cell lysate (WCL). (C and D) In vitro pulldown assay. Approximately 2 μg of the purified GST-MAAP2 protein and negative control GST protein were employed as baits to pull down ∼2 μg prey proteins; purified SNAP23 (C) or STX7 (D) using glutathione agaroses. Western blotting was performed for detection of GST-MAAP2 and control GST using anti-GST (C and D), for the detection of SNAP23 using anti-SNAP23 (C), and for the detection of STX7 using anti-STX7 (D). ∼200 ng of the bait and pray proteins were loaded as inputs. Asterisks indicate the major detected GST-MAAP and GST.

Article Snippet: An anti-V5 (#R960-25) was purchased from Invitrogen (Carlsbad, CA), and anti-STX7 antibodies were purchased from AbClonal (#A8057) and Bethyl Laboratories (#A304-512A; Montgomery, TX) for immunostaining and immunoblotting, respectively.

Techniques: In Vitro, Co-Immunoprecipitation Assay, Transfection, Immunoprecipitation, Magnetic Beads, Western Blot, Control, Purification, Negative Control

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Identification of the role of SNARE proteins in rAAV vector production through interaction with the viral MAAP

doi: 10.1016/j.omtm.2024.101392

Figure Lengend Snippet: Knockout of STX7 or SNAP23 increases rAAV vector secretion into the media, as well as total vector yields, during rAAV production (A and B) Generation of KO cell lines. (A) Western blotting. HEK293 cells were transduced with lentiviruses expressing STX7 -, SNAP23 -targeting, or scramble guide RNA (Scramble), followed by single-cell cloning for STX7-KO cell line generation. SNAP23-KO cell line was not single-cell cloned. Cells were analyzed for expression of STX7 or SNAP23, as indicated, using western blotting. β-actin serves as a loading control. (B) Cell viability. WT, Scramble, STX7-KO, and SNAP23-KO HEK293 cells were seeded in wells of six-well plates at equal cell number. Cells were trypsinized, and the viable cells were counted after staining with trypan blue at 24, 48, and 72 h, respectively. (C–F) Small-scale production of rAAV2 (C), rAAV1 (D), rAAV5 (E), and rAAV9 (F) in the gene knockout cells. WT, Scramble, STX7-KO, and SNAP23-KO HEK293 cells, as indicated, were transfected with pR2C2 (C), pR2C1 (D), pR2C5 (E), or R2C9 (F), together with pHelper and prAAV2. At 3 dpt, cells and media were harvested for subsequent treatments. DNase-digestion-resistant viral DNA was extracted from crude lysates of harvested cells (pellet) and media, respectively. Produced rAAV vectors in the pellet and media were quantified by qPCR using an mCherry probe. Left panel: Related bars represent total vector yields in vector genomic copies (vgc) in the pellet (cells), media and total (pellet plus media), respectively. Right panel: Bars indicate ratios of produced rAAV yield in the pellet (cells) vs. in media based on the data presented in the left panel. Means and SDs were calculated using data from three independent experiments ( n = 3). ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗∗ p < 0.0001; and ns, no significant difference.

Article Snippet: An anti-V5 (#R960-25) was purchased from Invitrogen (Carlsbad, CA), and anti-STX7 antibodies were purchased from AbClonal (#A8057) and Bethyl Laboratories (#A304-512A; Montgomery, TX) for immunostaining and immunoblotting, respectively.

Techniques: Knock-Out, Plasmid Preparation, Western Blot, Transduction, Expressing, Single Cell, Cloning, Clone Assay, Control, Staining, Gene Knockout, Transfection, Produced

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Identification of the role of SNARE proteins in rAAV vector production through interaction with the viral MAAP

doi: 10.1016/j.omtm.2024.101392

Figure Lengend Snippet: Large-scale production of rAAV5 in STX7-KO and SNAP23-KO HEK293 cells Scramble, STX7-KO, and SNAP23-KO HEK293 cells were transfected with pR2C5, pHelper, and prAAV2. (A) mCherry expression. Transfected cells were imaged for mCherry expression at 2 dpt by the ZOE Fluorescent Cell Imager (Bio-Rad). Representative images are shown. The intensity of mCherry expression of each group was displayed as fold changes relative to scramble control group. The relative fold changes of mCherry expression in the transfected cells were measured by ImageJ. (B) Titers of the purified rAAV5 vectors in the cells (pellet) and in the media. At 3 dpt, cells and media were harvested for vector purification. DNase-digestion-resistant viral DNA were extracted from the final purified vectors from the pellets and media and quantified by qPCR. Left panel: Data shown are total vector yields (vgc) in pellet, media or total (pellet plus media). Right panel: Bars indicate ratios of produced rAAV yield in the pellet (cells) vs. in the media based on the data presented in the left panel. (C) Transduction efficiency. HEK293 cells were transduced with rAAV5 purified from the cells and the media of the indicated HEK293 cell lines (x axis). Bars represent the intensity of luciferase activity of each group, shown as fold changes relative to the Scramble group (y axis). Means and SDs were calculated using data from three independent experiments ( n = 3). ∗ p < 0.05; ∗∗∗ p < 0.001; and ∗∗∗∗ p < 0.0001; and ns, no significant difference.

Article Snippet: An anti-V5 (#R960-25) was purchased from Invitrogen (Carlsbad, CA), and anti-STX7 antibodies were purchased from AbClonal (#A8057) and Bethyl Laboratories (#A304-512A; Montgomery, TX) for immunostaining and immunoblotting, respectively.

Techniques: Transfection, Expressing, Control, Purification, Plasmid Preparation, Produced, Transduction, Luciferase, Activity Assay

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Identification of the role of SNARE proteins in rAAV vector production through interaction with the viral MAAP

doi: 10.1016/j.omtm.2024.101392

Figure Lengend Snippet: A proposed model of the role of SNARE proteins in AAV egress AAV capsids produced in the nucleus are egressed through the nuclear pore complex (NPC), the endoplasmic reticulum (ER), and the Golgi apparatus, where a portion of the vectors are associated with EVs (marked with exosome marker CD63), on which MAAP2 is attached. , These EV-associated or -carried AAVs are routed to late endosomes (Rab7+) or MVB through the egress pathway of EVs as exosomes to secrete EV-AAVs. A portion of the late endosomes/MVBs are captured or recognized by v-SNARE proteins (e.g., STX7). STX7 interacts with t-SNARE proteins, e.g., SNAP23, for the fusion of late endosomes with lysosome, where SNAP23 makes up the heterodimeric t-SNAREs required for lysosome exocytosis, leading to the formation of lysosome/endosome hybrid for degradation of the virions. On the other hand, the EV-associated AAV can reach early endosomes (Rab5+) and then traffic to recycling endosomes (Rab11+), where they can be released as microvesicles. Knockout of SNARE expression can block the formation of a lysosome/endosome hybrid, resulting in less virion degradation and more virion release/secretion out of the plasma membrane.

Article Snippet: An anti-V5 (#R960-25) was purchased from Invitrogen (Carlsbad, CA), and anti-STX7 antibodies were purchased from AbClonal (#A8057) and Bethyl Laboratories (#A304-512A; Montgomery, TX) for immunostaining and immunoblotting, respectively.

Techniques: Produced, Marker, Knock-Out, Expressing, Blocking Assay, Clinical Proteomics, Membrane